1. Field of the Invention
The present invention relates to the determination of the presence or concentration of an analyte in a sample by visual or electronic means, using polarization based sensing techniques employing fluorescent sensing and reference molecules.
2. Description of the Related Art
A bibliography follows at the end of the Detailed Description of the Invention. The listed references are all incorporated herein by reference.
During the past ten years there have been remarkable advances in the technology for fluorescence sensing [1-7]. There has been extensive development of new fluorescent probes [8-10], and the introduction of time-resolved fluorescence to chemical sensing, which is referred to as lifetime-based sensing [11-13]. Additionally, the timescale of fluorescence has been extended from the nanosecond range to the microsecond range by the use of long-lifetime metal-ligand complexes [14-15].
New approaches to fluorescence sensing continue to appear. Recently a new approach to sensing has been developed which uses reference fluorophores in addition to the sensing fluorophores. The concept is to mix a sensing fluorophore which is sensitive to an analyte with a second fluorophore which is not sensitive to the analyte. One then measures the combined emission of the sensor and reference fluorophores, which can be used to determine the analyte concentration. This approach has been used with the long lifetime metal-ligand complexes (MLC) as the reference fluorophore, and a pH sensitive probe, to determine pH or pCO2 from the phase angle of the emission [16-17]. The present inventors have also used such mixtures to determine pH, calcium, and glucose concentrations. In our studies we used the low frequency modulation of the emission, rather than the phase angle, to determine the analyte concentration [18-21]. We showed that the low frequency modulation can be used to determine the fractional intensity of the nanosecond fluorophore, relative to that of the metal-ligand complex with its microsecond decay time.
In a recently published paper [22], the present inventors extended the idea of using a reference fluorophore to sensing based on anisotropy measurements. The concept is based on the additivity of anisotropy [23-25]. This rule states that the anisotropy for a mixture of fluorophores is the weighted average of the value for each fluorophore and their fractional contributions to the total intensity. Thus, an intensity change of the sensing fluorophore is transformed into a change in anisotropy or polarization by appropriate placement of polarizers. We developed sensing methods in which the reference was a fluorophore in a stretch-oriented film of polyvinyl alcohol. We used anisotropy-based sensing to measure pH using 6-carboxy fluorescein or the concentration of labeled protein in the sample.
However, there remains a need in the art for improved methods for determining the presence or concentration of an analyte using fluorescent reference and sensing molecules.
In one aspect, the present invention relates to a method for determining the presence or concentration of an analyte, comprising the steps of:
a) providing a fluorescent reference molecule and a fluorescent sensing molecule;
b) exposing said sensing molecule to an analyte to form a mixture, wherein said analyte is capable of changing the intensity of the fluorescence emitted by the sensing molecule in a concentration-dependent manner;
c) exposing said reference molecule and said. mixture to a radiation source which causes said reference and sensing molecules to emit fluorescence;
d) polarizing said emitted fluorescence through two different polarization axes which are substantially perpendicular to each other;
e) attenuating the emission from one of the polarization axes, if necessary, such that the intensities of the emissions through both axes are substantially equal; and
f) correlating the degree of attenuation with the presence or concentration of said analyte in said sample.
In another aspect, the present invention relates to a sensor for determining the presence or concentration of an analyte in a sample, which comprises:
a) a fluorescent reference molecule;
b) a fluorescent sensing molecule, wherein said analyte is capable of changing the intensity of the fluorescence emitted by the sensing molecule in a concentration-dependent manner;
c) optionally a radiation source which is capable of causing said reference and sensing molecules to emit fluorescence;
d) means for isolating said emitted fluorescence along two different polarization axes which are substantially perpendicular to each other; and
e) means for attenuating the emission from one of the polarization axes, such that the intensities of the emissions through both axes are substantially equal.